Primate immunodeficiency viruses target helper T-cells and macrophages/monocytes through binding of the viral envelope glycoprotein to a combination of CD4 and a chemokine receptor (CCR4 or CXCR5) on the surface of the host cells. Strikingly, infection results in rapid and sustained downregulation of CD4 and, to a lesser extent, the chemokine receptors. Downregulation of these viral co-receptors prevents superinfection, promotes virion release and interferes with the immune response, leading to the establishment of a robust infection. CD4 downregulation is so important to the life cycle of human immunodeficiency virus-1 (HIV-1) that two accessory proteins, Nef and Vpu, encoded in the viral genome are devoted to this task. Indeed, Nef and Vpu are critical for the progression from infection to AIDS, a fact that is best illustrated by the existence of long-term non-progressors that are infected with HIV-1 strains bearing inactivating mutations in the genes encoding these proteins. Therefore, pharmacologic or biologic perturbation of Nef and/or Vpu has the potential to prevent the pathogenic effects of HIV-1. To date, however, this potential has not been realized mainly because Nef and Vpu have no enzymatic activity and their mechanisms of action are insufficiently understood. In previous work, we made substantial progress towards elucidating the mechanism of CD4 downregulation by Nef. We found that Nef connects surface CD4 to both the endocytic and lysosomal targeting machineries, leading to efficient and sustained removal of CD4 from the host cells early during infection. The current project focuses on the mechanisms by which Vpu downregulates CD4 at later stages of infection. Vpu is a small transmembrane protein comprising a short luminal domain, a single transmembrane domain (TMD) and a cytosolic domain. The Vpu cytosolic domain simultaneously binds to the CD4 cytosolic tail and the SCF-beta-TrCP E3 ubiquitin ligase complex, causing CD4 ubiquitination and its subsequent targeting for degradation by the proteasome. Our studies revealed the following novel aspects of this process: (i) degradation involves at least some components of the cellular ER-associated degradation (ERAD) machinery, including the VCP-UFD1L-NPL4 dislocase complex; (ii) CD4 ubiquitination depends on not only lysine but also serine and threonine residues in the CD4 tail; (iii) Vpu mediates CD4 retention in the ER in addition to targeting to ERAD. The multiple levels at which Vpu engages the cellular quality control mechanisms underscore the importance of ensuring profound suppression of CD4 to the life cycle of HIV-1. This past year we showed that the transmembrane domains (TMD) of both Vpu and CD4 are required for Vpu-induced CD4 downregulation. We found that Trp22 in the Vpu TMD and Gly415 in the CD4 TMD are critical for Vpu-induced targeting of CD4 to endoplasmic reticulum (ER)-associated degradation (ERAD). Vpu Trp22 promotes CD4 polyubiquitination and subsequent recruitment of the VCP-UFD1L-NPL4 dislocase complex. In the presence of a Vpu Trp22 mutant, CD4 remains integrally associated with the ER membrane, suggesting that dislocation from the ER into the cytosol is impaired. CD4 Gly415, on the other hand, contributes to CD4-Vpu interactions. We also identified two residues, Val20 and Ser23, in the Vpu TMD that mediate retention of CD4 in the ER. These findings highlight the exploitation of several TMD-mediated mechanisms by HIV-1 Vpu in order to downregulate CD4 and thus promote viral pathogenesis.